in the method used to separate

the antigen-antibody combination from the unbound

Serology/Immunology 591

antigen, and in the standardization. The majority of

current methods use 125I (radioactive iodine) or 3

H

(tritium, the radioactive isotope of hydrogen) as the

labels. For separation of antigen-antibody combination,

charcoal coated with dextran is used. The dextran acts

as a molecular sieve that passes only unbound antigen

molecules for retention by the charcoal, the antigenantibody combinations are too large to cross the dextran

coating. After centrifuging, the relatively dense charcoal

grains with their adsorbed antigen molecules will be

packed at the bottom of the tube, and the supernatant

containing the antigen-antibody combinations can be

separated. Measurement of the ratio of radioactivities

of the two components completes the assay. A more

sophisticated method of precipitating the antigenantibody combination is to add a second antibody

prepared to react with the protein of the first antibody,

usually a gamma globulin. The resultant complex can then

be separated either by centrifugation or cellulose acetate

filters. Standardization can be done as described in the

description of general principles above.

The RIA technique promises to provide reliable data

by relatively simple methods about biological substances

that present considerable analytical problems when more

orthodox procedures are used. In practice, of course, RIA

has its own sources of error. These include:

¾ Lability of compound analyzed

¾ Antibody cross reaction with related antigens

¾ Interfering substances in the sample, e.g. urea and

bilirubin

¾ Poor pipetting technique (good pipetting technique is

critical, because of the very small volumes)

¾ Contamination of equipment from extraneous

radioactive materials

¾ Change in the antigen’s chemical or immunological

identity owing to the process of adding radioactive

label to the antigen.

The RIA methods measure the amounts of particular

molecular structures, not their biological activity.

Measurement of Radioactivity

The radioactive atoms used as labels produce different

types of emitted radiation. 125I emits short-wavelength,

high-energy gamma rays; 3

H (tritium) produces betatype radiation, which is actually high-speed particles,

positively or negatively charged electrons. Gamma rays

are detected by a so-called scintillation counter, which

consists of a large sodium iodide crystal that contains

thallium as an activator. The crystal is in close contact

with a photomultiplier tube; and when an emitted

quantum of gamma radiation strikes a sodium iodide

molecule in the crystal lattice, it produces a photon of

light energy. This light is picked up and amplified by the

assopifeted photomultiplier tube and converted to a pulse

of electrical energy. The number of pulses is proportional

to the quantity of radioactive material in the sample, the

power or energy of the pulse is determined by the energy

of the original gamma ray. The scintillation counter

incorporates “discriminators,” which pass through only

those pulses whose energy levels correspond to those

of the gamma radiation emitted from the particular

radioactive atom whose detection is required. Finally, the

scintillation counter uses a sealer to count the number

of pulses arriving in a preset time or to determine the

time required for a preset number of pulses to occur. To

detect beta particles, which have less energy than gamma

radiation is used.

The preparation of serology reagents and anti-sera is

much too complex and beyond the scope of this book.

It is, therefore, advised that ready-made kits available

commercially be used. Basic principles are mentioned.

Product insert must, however, be read and followed strictly.

LIQUID HANDLING SYSTEMS

Pipetting Basics

Human beings are creature of habits. We often seek

stability and continuity and are very much wary of damage.

Pipetting in history was carried out most exclusively

by suction using a glass pipette. However, inspection,

evaluation and subsequent changes are necessary for

growth and improvement. Though these methods were

convenient and economical, they lack accuracy and precision. Secondly aspirating small volumes of liquid using a

glass pipette is not possible.

Classification of Pipettes

There are many ways of classifying pipettes:

Based on the Material

Glass pipettes

It is a traditional old pipette made of long glass tube scaled

for different volumes by a marking on its surface. The

principle of aspiration of the liquid is by suction. Though

this method is convenient and economical, it lacks accuracy

and precision.

Plastic pipette

Made of total plastic components and parts. It is the most

commonly used pipette. Some pipettes are difficult to

592 Concise Book of Medical Laboratory Technology: Methods and Interpretations calibrate and are not fully autoclavable. Dissembling and

assembly is not possible in most of the pipettes. Also both

variable and fixed volume is not in one pipette as compared

to “New Third Generation Pipettes”. The principle of

operation is by suction.

Metal pipettes

They are called as new generation pipettes and are being

increasingly used commonly. These pipettes are made

of anodized aluminum and the piston made of stainless

steel. These come with detachable controllers for variable

and fixed volumes with digital volume setting.

Based on Function

Fixed and variable pipette

These may be plastic or partial metal pipettes but serving

only one function. They can either be used for aspiration

of fixed volume of liquids or a specific range of volumes.

Combined pipettes

These pipettes offer the flexibility and user friendliness

of both variable and fixed volume options in the same

pipette.

Pipetting Techniques

The first step in pipetting is to choose the pipetting mode

best suited to the type of work. These pipetting modes are:

Forward Pipetting

It is the standard technique for pipetting aqueous liquids.

1. Press the operating button to the first step.

2. Dip the tip into the solution to a depth of 1 cm and

slowly release the button. Withdraw the tip from

liquid, touching it against the edge of the reservoir to

remove excess liquid.

3. Dispense the liquid into the receiving vessel by gently

pressing the operating button to the first step. After

one second, press the button down to the second stop.

This action will empty the tip. Remove the tip from the

vessel, sliding it along the wall of the vessel.

4. Release the operating button to the ready position.

Reverse Pipetting

This technique is used for pipetting solutions of high

viscosity or a tendency to foam. This method is also

recommended for dispensing small volumes.

1. Press the operating button to the second stop.

2. Dip the tip into the solution to a depth of 1cm and

slowly release the button. This action will fill the tip.

Withdraw the tip from the liquid, touching it against

the edge of the reservoir to remove excess liquid.

3. Dispense the liquid into the receiving vessel by

pressing the button gently and steadily down to the

first stop. Hold the button in this position. Some

liquid will remain in the tip, and this should not be

dispensed.

4. The liquid remaining in the tip can be pipetted back

into the original solution or thrown away with the tip.

5. Release the operating button to the ready position.

Repetitive Pipetting

This technique is intended for repeated pipetting of the

same volume.

1. Press the operating button down to the second stop.

2. Dip the tip into solution to a depth of 1cm and slowly

release the operating button. Withdraw the tip from

the liquid, touching it against the edge of reservoir to

remove excess liquid.

3. Dispense the liquid in the receiving vessel by gently

pressing the operating button to the first stop. Hold

the button in this position. Some liquid will remain

in the tip and this should not be dispensed.

4. Continue pipetting by repeating steps 2 and 3.

Whole Blood Pipetting

Use forward technique steps 1 and 2 to fill the tip with

blood. Wipe the tip carefully with a dry clean cloth.

1. Dip the tip into the reagent and press the operating

button down to the first stop. Make sure the tip is well

below the surface.

2. Release the button slowly to the ready position. This

action will fill the tip with the reagent. Do not remove

the tip from the solution.

3. Press the button down to the first stop and release

slowly. Repeat this process until the interior wall of

the tip is clear.

4. See 3.

5. Press the button down to the second stop and completely empty the tip.

6. Release the operating button to the ready position.

Proper Pipetting Skills

1. Warming up the pipette mechanism: The pipette

mechanism should always be warmed up before

starting by gently pressing and releasing the plunger

15–20 times.

2. Pre-wet the pipette tip: Aspirate and expel and amount

of the sample liquid at least 2–3 times before aspirating